NMR EVIDENCE FOR THE PARTICIPATION OF A LOW-BARRIER HYDROGEN-BOND IN THE MECHANISM OF DELTA(5)-3-KETOSTEROID ISOMERASE

Delta(5)-3-Ketosteroid isomerase (EC 5.3.3.1) promotes an allylic rear rangement involving intramolecular proton transfer via a dienolic inte rmediate. This enzyme enhances the catalytic rate by a factor of 10(10 ). Two residues, Tyr-14, the general acid that polarizes the steroid 3 -carbonyl group and facilitates enolization, and Asp-38 the general ba se that abstracts and transfers the 4 beta-proton to the 6 beta-positi on, contribute 10(4.7) and 10(5.6) to the rate increase, respectively. A major mechanistic enigma is the huge disparity between the pK(a) va lues of the catalytic groups and their targets, Upon binding of an ana log of the dienolate intermediate to isomerase, proton NMR detects a h ighly deshielded resonance at 18.15 ppm in proximity to aromatic proto ns, and with a 3-fold preference for protium over deuterium (fractiona tion factor phi = 0.34), consistent with formation of a short, strong (low-barrier) hydrogen bond to Tyr-14, The strength of this hydrogen b ond is estimated to be at Least 7.1 kcal/mol. This bond is relatively inaccessible to bulk solvent and is pH insensitive. Low-barrier hydrog en bonding of Tyr-14 to the intermediate, in conjunction with the prev iously demonstrated tunneling contribution to the proton transfer by A sp-38, provide a plausible and quantitative explanation for the high c atalytic power of this isomerase.